The present invention relates generally to immunological carrier systems. More particularly, the invention pertains to leukotoxin-antigen chimeras which demonstrate enhanced immunogenicity as compared to the immunogenicity of the antigen alone.
Subunit vaccines are vaccines which are devoid of intact pathogen cells. These vaccines are usually composed of substantially purified antigens. Such vaccines are generally preferable to compositions which use attenuated or inactivated pathogens. However, many subunit vaccines which include proteins, such as peptide hormones and bacterial and viral antigens, require the help of a carrier protein in order to elicit a strong immune response. This is especially true for small proteins or endogenous substances, such as hormones, which are poorly immunogenic.
The carrier serves to non-specifically stimulate T helper cell activity and to direct the antigen to the antigen presenting cell, where the antigen is processed and presented at the cell surface in the context of molecules of the major histocompatibility complex (MHC).
Several carrier systems have been developed for this purpose. For example, small peptide antigens are often coupled to protein carriers such as keyhole limpet haemocyanin (Bittle, J. L., et al., Nature (1982) 298:30-33), tetanus toxoid (Muller, G., et al., Proc. Natl. Acad. Sci. U.S.A. (1982) 79:569-573), ovalbumin, and sperm whale myoglobin, to produce an immune response. However, carriers may elicit strong immunity not relevant to the peptide antigen and this may inhibit the immune response to the peptide vaccine on secondary immunization (Schutze, M. P., et al, J. Immun. (1985) 135:2319-2322).
Antigen delivery systems have also been based on particulate carriers. For example, preformed particles have been used as platforms onto which antigens can be coupled and incorporated. Systems based on proteosomes (Lowell, G. H., et al., Science (1988) 240:800-802), immune stimulatory complexes (Morein, B., et al., Nature (1984) 308:457-460), and viral particles such as HBsAg (Neurath, A. R., et al., Mol. Immunol. (1989) 26:53-62) and rotavirus inner capsid protein (Redmond, M. J., et al., Mol. Immunol. (1991) 28:269-278) have been developed.
Other carrier systems have been devised using recombinantly produced chimeric proteins that self assemble into particles. For example, the yeast retrotransposon, Ty, encodes a series of proteins that assemble into virus like particles (Ty-VLPs; Kingsman, S. M., and A. J. Kingsman Vacc. (1988) 6:304-306). Foreign genes have been inserted into the TyA gene and expressed in yeast as a fusion protein. The fusion protein retains the capacity to self assemble into particles of uniform size.
Other chimeric protein particles have been examined such as HBsAg, (Valenzuela, P., et al., Bio/Technol. (1985) 3:323-326; U.S. Pat. No. 4,722,840; Delpeyroux, F. N., et al., Science (1986) 233:472-475), Hepatitis B core antigen (Clarke, B. E., et al., Vaccines 88 (Ed. H. Ginsberg, et al., 1988) pp. 127-131), Poliovirus (Burke, K. L., et al., Nature (1988) =:81-82), and Tobacco Mosaic Virus (Haynes, J. R., et al., Bio/Technol. (1986) 4:637-641). However, these carriers are restricted in their usefulness by virtue of the limited size of the active agent which may be inserted into the structural protein without interfering with particle assembly.
Gene fusions provide a convenient method for the production of chimeric proteins. The expression of chimeric proteins affords an efficient means of linking a carrier protein to a desired antigen.
Pasteurella haemolytica produces a cytotoxin which is a leukotoxin. See, e.g. Gentry et al. Vet. Immunology and Immunopathology (1985) 9:239-250; Shewen, P. E., and Wilkie, B. N. Infect. Immun. (1987) 55:3233-3236. The gene encoding this cytotoxin has been cloned and expressed in bacterial cells. Lo et al. Infect. Immun. (1985) 50:667-671; U.S. Pat. No. 5,055,400. The leukotoxin has been used as an antigen in vaccine formulations to fight shipping fever pneumonia in livestock (See, e.g. U.S. Pat. No. 4,957,739) as well as to produce chimeric molecules for use in vaccines against shipping fever (see, e.g. International Publication No. WO 92/03558, published Mar. 5, 1992; and U.S. Pat. No. 5,028,423). However, the use of leukotoxin as a carrier molecule to increase the immune response of antigens associated therewith has not heretofore been described.
The present invention is based on the construction of novel gene fusions between the P. haemolytica leukotoxin gene and a nucleotide sequence encoding a selected antigen. These constructs produce a chimeric protein that displays enhanced immunogenicity when compared to the immunologic reaction elicited by administration of the antigen alone.
In one embodiment, the present invention is directed to an immunological carrier system comprising an immunogenic chimeric protein. The chimeric protein comprises a leukotoxin polypeptide fused to a selected antigen, whereby the leukotoxin portion of the chimeric protein acts to increase the immunogenicity of the antigen. In particularly preferred embodiments, the selected antigen is somatostatin (SRIF), gonadotropin releasing hormone (GnRH) or rotavirus viral protein 4 (VP4).
Also disclosed are vaccine compositions comprising the chimeric proteins and a pharmaceutically acceptable vehicle and methods of using the same.
In another embodiment, the subject invention is directed to DNA constructs encoding the chimeric proteins. The DNA constructs comprise a first nucleotide sequence encoding a leukotoxin polypeptide operably linked to a second nucleotide sequence encoding the selected antigen.
In yet another embodiment, the subject invention is directed to expression cassettes comprised of (a) the DNA constructs above and (b) control sequences that direct the transcription of the construct whereby the constructs can be transcribed and translated in a host cell.
In another embodiment, the invention is directed to host cells transformed with these expression cassettes.
Another embodiment of the invention provides a method of producing a recombinant polypeptide. The method comprises (a) providing a population of host cells described above and (b) growing the population of cells under conditions whereby the polypeptide encoded by the expression cassette is expressed.
These and other embodiments of the present invention will readily occur to those of ordinary skill in the art in view of the disclosure herein.